BURULI12.COVER.CAGDiagnosis of Mycobacterium ulcerans disease
E D I T E D B Y : F R A N Ç O I S E P O R T A E L S P A U L J O H N S O N W A Y N E M . M E Y E R S
WHO/CDS/CPE/GBUI/2001.4 DISTRIBUTION: GENERAL
ORIGINAL: ENGLISH
A M A N U A L F O R H E A L T H C A R E P R O V I D E R S
World Health Organization
This manual was published thanks to financial support from:
The Association Française Raoul Follereau (AFRF), France is an NGO dedicated to leprosy control in 31 countries worldwide. It also supports six research projects on leprosy, including the genome sequencing of Mycobacterium leprae. Long before the first International Conference on Buruli Ulcer Control and Research, Yamoussoukro, Côte d’Ivoire, 1998, AFRF had taken up the new challenge of the health and social problems caused by Buruli ulcer, working in Benin and Côte d’Ivoire since 1996. The Association also provides financial
assistance to research activities on the genome sequencing of Mycobacterium ulcerans and on the drug treatment of the disease. It is now considering supporting other countries, starting with Ghana. AFRF is committed to mobilizing the international support needed to meet the challenges posed by Buruli ulcer. For more information, visit the AFRF website: http://www.raoul-follereau.org
ANESVAD, Spain is an NGO that has been working against leprosy and implementing health, social and educational projects in 28 of the poorest developing countries for over 30 years. Currently it counts on the support of over 135 000 partners and collaborators in Spain. It has recently begun work on Buruli ulcer in
Côte d’Ivoire, carrying out programmes to detect the disease at an early stage and undertaking prevention, surgical treatment, training of specialized medical staff and social awareness campaigns, with the aim of limiting the impact of Buruli ulcer. For more information, visit the ANESVAD website: http://www.anesvad.org
Médecins Sans Frontières (MSF) is an international humanitarian aid organization that provides emergency medical assistance to populations in danger in more than 80 countries. MSF Luxembourg has been involved in Buruli ulcer control activities in Benin since 1997. MSF has upgraded the Lalo Health Centre with surgical and laboratory facilities to improve the care of patients. Apart from surgical activities, other key activities include
health education in affected communities, case-finding and training of health care providers, teachers and traditional healers. In terms of Buruli ulcer research, MSF is collaborating with the Institute of Tropical Medicine, Antwerp, Belgium. For more information, visit the MSF Luxembourg's website at: http://www.msf.lu
The Nippon Foundation, Japan is a private grant-making foundation whose activities cover social welfare, public health, volunteer support and overseas assistance. Since 1975 it has been working through the Sasakawa Memorial Health Foundation to aid WHO in its fight to eliminate leprosy. Starting in 1998, The Nippon Foundation also began providing financial support to the WHO Global Buruli Ulcer Initiative. The Foundation, in tandem
with WHO and several academic institutions, is currently exploring options for improved surgical management of the disease. Finally, it is also collaborating with WHO, AFRF and other partners to find a drug treatment for Buruli ulcer. For more information, visit The Nippon Foundation’s website at: http://www.nippon-foundation.or.jp
BuruliUlcer Diagnosis of Mycobacterium ulcerans disease
E D I T E D B Y :
PROFESSOR FRANÇOISE PORTAELS Department of Microbiology Inst itute of Tropical Medicine Antwerp, Belgium
ASSOCIATE PROFESSOR PAUL JOHNSON Department of Infect ious Diseases Austin and Repatr iat ion Medical Centre Heidelberg, Melbourne, Austral ia
DOCTOR WAYNE M. MEYERS Divis ion of Microbiology Armed Forces Inst itute of Pathology Washington, DC, United States of America
A M A N U A L F O R H E A L T H C A R E P R O V I D E R S
World Health Organization
© World Health Organization, 2001
This document is not a formal publication of the World Health Organization (WHO), and all rights are reserved by the Organization. The document may, however, be freely reviewed, abstracted, reproduced or translated, in part or in whole, but not for sale or for use in conjunction with commercial purposes. The views expressed in documents by named authors are solely the responsibility of those authors.
Design: Gilles Lasseigne – Layout: Bruno Duret
Acknowledgements
With special thanks to Rosemary Bell, France, and John Hayman, Monash University, Australia.
Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 Illustrations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Chapter 1. Clinical diagnosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 Chapter 2. Biosafety and record-keeping in the laboratory . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 Chapter 3. Collection and transport of clinical specimens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19 Chapter 4. Secondary bacterial infection in M. ulcerans disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 Chapter 5. Microbiological methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27 Chapter 6. Histopathological methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37
Annex 1. Flow chart for the laboratory diagnosis of M. ulcerans disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48 Annex 2. Laboratory request form . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49 Annex 3. Laboratory report form . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51 Annex 4. Preparation of culture media . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52 Annex 5. Microbiological staining techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54 Annex 6. Histopathological staining techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59 Annex 7. Decontamination methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68 Annex 8. M. ulcerans culture with BACTEC 460 TB instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71 Annex 9. Biochemical and culture tests used to identify mycobacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72 Annex 10. Polymerase chain reaction (PCR) protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78 Annex 11. Manufacturers’ addresses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84 Annex 12. Work of WHO on Buruli ulcer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85 Annex 13. Some research institutions involved in Buruli ulcer activities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87
Contents
Prof. Ohene Adjei, Department of Microbiology, School of Medical Sciences, University of Science and Technology, Kumasi, Ghana / Prof. Bernard Carbonnelle, Laboratoire de Bactériologie, Centre Hospitalier Universitaire d’Angers, Angers, France / Prof. Patience Mensah, Bacteriology Unit, Noguchi Memorial Institute for Medical Research, University of Ghana, Accra, Ghana / A/Prof. Paul Johnson, Department of Infectious Diseases, Austin and Repatriation Medical Centre, Heidelberg, Melbourne, Australia / Dr Henri Kouakou, Institute Raoul Follereau, Adzope, Côte d’Ivoire / Dr Wayne M. Meyers, Division of Microbiology, Armed Forces Institute of Pathology, Washington, DC, USA / Prof. Françoise Portaels, Department of Microbiology, Institute of Tropical Medicine, Antwerp, Belgium / Dr Kingsley Asiedu, Communicable Diseases Control, Prevention and Eradication, World Health Organization, Geneva, Switzerland
Annex 14. Some nongovernmental organizations and others involved in Buruli ulcer activities . . . . . . . . . . . . . . . . . . . . 88 Annex 15. Members of the WHO Advisory Group on Buruli ulcer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89 Annex 16. Suggested reading . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90
Table 1 Differential diagnoses of various forms of Buruli ulcer Table 2 Disinfectants recommended for use in laboratories studying M. ulcerans Table 3 Specimen collection and laboratory methods for each care-level Table 4 Phenotypic characteristics of M. ulcerans and related species Table 5 Characteristics of the different geographical subgroups of M. ulcerans Table 6 In vitro susceptibility of M. ulcerans to antimycobacterial drugs Table 7 Preparation of inhibitory agents Table 8 Urease activity
Contributors
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Preface
This manual is to assist health care providers and laboratory scientists to diagnose Mycobacterium ulcerans disease (Buruli ulcer). The manual aims to achieve a better understanding of the clinical presentation and its diagnosis. The methods described are tailored to various levels of care and available resources to improve the diagnosis and surveillance of the disease.
Please note: This manual is not intended to serve as a standard of laboratory methods. It is not a replacement for textbooks on laboratory work. Adherence to it will not ensure a successful outcome in every case, nor should it be construed as including all proper methods of laboratory diagnosis or excluding other acceptable methods aimed at the same results. Ultimate judgement regarding a particular method must be made by the health care provider or laboratory scientist in the light of the clinical findings in the patient and the available options for diagnosis.
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Illustrations Fig. 1 World map showing distribution of Buruli ulcer (WHO) Fig. 2 Papule (John Hayman) Fig. 3 Nodule (Mark Evans) Fig. 4 Plaque (Mark Evans) Fig. 5 Oedematous forms (May Smith and Kingsley Asiedu) Fig. 6 Ulcers (May Smith and Mark Evans) Fig. 7 Osteomyelitis (Giovanni Batista Priuli) Fig. 8 Contractures (Marcel Crozet) Fig. 9 Hypertrophic scar (Pius Agbenorku) Fig. 10 Squamous cell carcinoma following Buruli ulcer
(Mark Evans) Fig. 11 Differential diagnosis (Wayne Meyers) Fig. 12 Containers for specimens (Paul Johnson) Fig. 13 Swabbing technique (May Smith) Fig. 14 Mouse tail inoculation (Bernard Carbonnelle) Fig. 15 Culture characteristics of African and Australian
M. ulcerans strains (Françoise Portaels) Fig. 16 Polymerase chain reaction results (Paul Johnson) Fig. 17 Section of surgically resected nodule of M. ulcerans
disease (John Hayman) Fig. 18 Microscopic section of a nodule (AFIP) Fig. 19 Skin and subcutaneous tissue from centre of a
non-ulcerated lesion (AFIP courtesy Wayne Meyers) Fig. 20 Necrotic base of Buruli ulcer (AFIP courtesy
Wayne Meyers) Fig. 21 Severe vasculitis in subcutaneous tissue lesion
(John Hayman) Fig. 22 Fat cell ghosts and vasculitis (AFIP courtesy
Wayne Meyers) Fig. 23 Ziehl-Neelsen stain of a section parallel to that
of Figure 18 (AFIP courtesy Wayne Meyers) Fig. 24 Subcutaneous tissue from the edge of a Buruli ulcer
(AFIP courtesy Wayne Meyers) Fig. 25 Masses of AFB in the base of a Buruli ulcer
(AFIP courtesy Wayne Meyers) Fig. 26 Biopsy specimen from the edge of a Buruli ulcer
(AFIP courtesy Wayne Meyers) Fig. 27 Subcutaneous tissue from margin of a Buruli ulcer
(AFIP courtesy Wayne Meyers)
Fig. 28 Early healing of a Buruli ulcer in the organizing phase (AFIP Courtesy Wayne Meyers)
Fig. 29 Delayed hypersensitivity granuloma in healing Buruli ulcer (AFIP)
Fig. 30 Advanced stage of healing Buruli ulcer (AFIP courtesy Wayne Meyers)
Fig. 31 Lymphadenopathy in Buruli ulcer (AFIP courtesy Wayne Meyers)
Fig. 32 Necrotic lymphadenitis in a lymph node proximal to a Buruli ulcer (AFIP courtesy Wayne Meyers)
Fig. 33 X-ray of the leg showing destruction of the bone (Giovanni Battista Priuli)
Fig. 34 Osteomyelitis of tibia showing necrosis of the marrow (AFIP courtesy Wayne Meyers)
Fig. 35 Osteomyelitis of tibia with masses of AFB in necrotic marrow (AFIP courtesy Wayne Meyers)
Fig. 36 Osteomyelitis of tibia showing necrosis of marrow (AFIP courtesy Wayne Meyers)
Fig. 37 Ziehl-Neelsen stained smear from a Buruli ulcer (Françoise Portaels)
Fig. 38 Fluorochrome stained smear showing AFB (Wellcome Trust courtesy of AM Emmerson)
Fig. 39 Section of tissue from a Buruli ulcer patient stained by the Harris’ hematoxylin-eosin method showing panniculitis (AFIP courtesy Wayne Meyers)
Fig. 40 Section of a lymph node from a Buruli ulcer patient stained by the Ziehl-Neelsen method showing AFB (AFIP courtesy Wayne Meyers)
Fig. 41 Histopathological section of a phaeomycotic cyst in skin stained by Grocott methenamine-silver method (AFIP courtesy Wayne Meyers)
Fig. 42 Gram-stain of tissue infected by Rhodococcus sp. (AFIP courtesy Wayne Meyers)
Fig. 43 Scotochromogenic, photochromogenic, or non- photochromogenic characteristics of mycobacteria (Françoise Portaels)
Fig. 44 Catalase activity (Françoise Portaels)
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In 1998, the World Health Organization (WHO) established the Global Buruli Ulcer Initiative (GBUI) in response to the growing spread and impact of Buruli ulcer, Mycobacterium ulcerans disease. The disease exists or has been suspected in at least 31 countries (Fig. 1). The primary objectives of the GBUI are: to raise awareness of the disease, to mobilize support for affected countries, to promote and to coordinate research activities and to coordinate the work of nongovernmental organizations (NGOs) and other partners. A summary of the achievements of the GBUI is presented in Annex 12.
In 1897, Sir Albert Cook in Uganda described skin ulcers consistent with Buruli ulcer but he did not publish these cases in the medical literature. In 1948, MacCallum et al. published the first confirmed cases of the disease. These patients were in Australia. The disease was called Bairnsdale ulcer after the main town in the original endemic region. In south- eastern Australia, the disease is still often referred to as Bairnsdale ulcer but, in parts of Africa, it is called “Buruli ulcer”, the name coming from a county in Uganda where large numbers of cases were reported in the 1950s.
It is called “Buruli ulcer”, the name coming from a county in Uganda where cases were reported in the 1950s.
Introduction
Figure 1 Worldwide distribution of Mycobacterium ulcerans disease
Note: Shaded areas do not represent the extent of the problem but indicate only those countries where the disease has been reported or suspected.
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Epidemiology and transmission After tuberculosis and leprosy, Buruli ulcer is the most common mycobacterial infection of humans. It is caused by Mycobacterium ulcerans. The disease often occurs in people who live or work close to rivers and stagnant bodies of water. Changes in the environment, such as the construction of irrigation systems and dams, seem to have played a role in the resurgence of the disease. The mode of transmission is not known, but recent evidence suggests that aquatic insects (Naucoris and Dyplonychus species) may be involved. Trauma to contaminated skin sites appears to be the means by which the organism enters the body. There is little proven evidence of transmission from person to person. No racial or social group is exempt. Infection with the human immunodeficiency virus (HIV) is not a known risk factor. The disease is more severe in impoverished inhabitants of remote rural areas. About 70% of those affected are children under the age of 15 years. Mortality due to the disease is low, but morbidity is high. Complications include contracture deformities, amputation of limbs, and involvement of the eye, breast and genitalia. In some localities 20–25% of those with healed lesions are left with disabilities that have a long- term social and economic impact. The current economic and social burden imposed by Buruli ulcer is enormous. In Ghana, the average cost of treatment per patient is estimated to be US$ 780. The prevalence of the disease is not accurately known. In Côte d’Ivoire, over 15 000 cases were recorded between 1978 and 1999. Prevalence rates have been estimated at 16% in some communities in Côte d’Ivoire and at 22% in a community in Ghana. In Benin, nearly 4 000 cases were
reported between 1989 and 1999. In Ghana, a survey conducted in 1999 identified over 6 000 cases and showed for the first time that all 10 regions of the country are affected. Cases have also been reported in Burkina Faso, Togo, Guinea and other West African countries. A few cases have been reported in non-endemic areas in North America and Europe as a sequel to international travel. Lack of familiarity with Buruli ulcer has frequently resulted in significant delays in the diagnosis and treatment of these cases.
The causative organism Mycobacterium ulcerans is a slow growing environmental mycobacterium. It is an acid-fast micro-organism that grows on common mycobacteriological media, e.g. Löwenstein- Jensen (L-J) medium. It grows best at low temperatures (30–32 °C), at lower than atmospheric oxygen tension (pO2 < 2.5 kPa) and within a pH range of 5.4–7.4. A positive culture requires incubation for 6 to 8 weeks (or longer) under appropriate conditions.
Toxin A toxin that causes tissue necrosis has been known for some time. Recently, one such compound—a polyketide- derived macrolide called mycolactone—has been identified and its chemical structure established. The toxin has both cytotoxic and local immunosuppressive properties. Injection of the purified toxin into experimental animals causes changes in subcutaneous fat similar to those seen in Buruli ulcers. This is the first macrolide known to be produced by a human pathogen and the only macrolide identified in the genus Mycobacterium.
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Pathogenesis Once introduced into the subcutaneous tissue the organism proliferates and elaborates a toxin that has affinity for fat cells. The resulting necrosis then provides a favourable milieu for further proliferation of the organism. During the necrotic phase, there is very little or no cellular immune response and the burulin skin test is negative. By an unknown mechanism, either the toxin may be neutralized or the organism may cease to proliferate or to produce toxin. Healing seems to begin when the host develops cell- mediated immunity, at which time the burulin skin test may become positive. The inflammatory cells then destroy the etiological agent (M. ulcerans) and the disease subsides with scarring. Bones may be affected by direct spread from the lesion or as a result of M. ulcerans bacteraemia. In contrast to other pathogenic mycobacteria, which are facultative intracellular parasites of macrophages, M. ulcerans occurs primarily as extracellular microcolonies.
Clinical spectrum of the disease Clinically the disease manifests as papules, nodules, plaques, oedematous forms and ulcers. The disease may be active (ongoing infection) or inactive (previous infection with characteristic depressed stellate scars with or without other sequelae). A new case is a patient with no previous history of, or treatment for, Buruli ulcer. A recurrent case is a patient presenting within one year with a further lesion at the same or a different site. Recurrence rates vary from 16% for patients presenting early to 28% for patients presenting late. Recurrence at the same site may be due to inadequate excision. Recurrence at a different site may be due to haematogenous or lymphatic spread.
Diagnosis Clinical: In a known endemic area, an experienced person can make the diagnosis of Buruli ulcer on clinical grounds. The following clinico-epidemiological features are important diagnostic clues: 1) the patient lives in or has travelled to a known endemic area; 2) most patients are children under 15 years of age; 3) about 85% of lesions are on the limbs; 4) lower limb lesions are twice as common as upper limb lesions.
Laboratory: Any two of the following findings are required to positively diagnose Buruli ulcers: 1) acid-fast bacilli in a smear stained by the Ziehl-Neelsen (ZN)
technique; 2) positive culture of M. ulcerans (but this requires 6–8 weeks
or longer); 3) histopathological study of excisional biopsy specimen (result
available rapidly); 4) positive polymerase chain reaction (PCR) for DNA from
M. ulcerans.
Treatment Drug treatment: Several antimycobacterial agents have in vitro activity against the causative organism but no single agent has been proven to be regularly useful in the treatment of the disease. Agents used include rifampicin, rifabutin, clarithromycin, azithromycin, streptomycin and amikacin. Combinations of agents have been used, with apparently varying success. Drug treatment alone, even with combi- nations of drugs, is usually ineffective when there is an established, progressing lesion. Research into drug treatment is a priority.
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Surgical treatment: This is accepted as the current definitive treatment. Limiting factors include: 1) inadequate surgical facilities; 2) need for prolonged stay in hospital; 3) high treatment costs; 4) recurrence after surgical treatment (rates of 16% to 28%); 5) the risk of transmission of infections such as HIV. Other adjuncts to treatment include heat and hyperbaric
oxygen, which have not been definitively proven and may be impractical in developing countries.
Control and prevention Community control strategies are currently limited by a lack of knowledge regarding the source of infection and the mode of transmission. The current standard treatment is surgery. Expert opinion is that early surgical management leads to improved results and resolution that are both cost saving. Early treatment is best promoted by an effective village-based surveillance programme. Current attitudes and beliefs may stigmatize and create fear in the affected individuals thereby delaying early and effective treatment. Educational materials should dispel such misinformation
and focus on early detection and surgery. Minor surgery (e.g., nodulectomies) may be performed at the local level.
What you should do The current control strategy promoted by the Global Buruli Ulcer Initiative consists of: • health education and staff training in the communities
most affected; • strengthening the health care capacity in endemic areas
by upgrading surgical facilities, ensuring adequate treatment supplies and improving laboratories;
• surgical training to enable other health workers (e.g. nurses, medical assistants) to perform effective minor surgery;
• community-based surveillance to improve early detection and rapid referral for treatment in collaboration with disease control programmes such as those for leprosy and dracunculiasis;
• adoption of educational material adapted to the needs of each country;
• developing successful motivational strategies; • rehabilitation of those already deformed by the disease.
Key points 1) About 70% of those infected with Buruli ulcer are children under 15 years old.
2) In Ghana the average cost to treat Buruli ulcer is over US$ 780 per person.
3) The accepted current treatment for Buruli ulcer is usually surgery.
Clinical diagnosis
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Non-ulcerative forms I Ulcerative forms I Bone involvement I Complications and sequelae I Differential diagnosis
Chapter 1
Clinical diagnosis
Credit: WHO
Clinical diagnosis
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Clinical diagnosis This chapter will assist you to recognize different forms of Mycobacterium ulcerans disease and to diagnose the condition irrespective of the stage at which it presents.
O b j e c t i v e s
Always consider the diagnosis of Mycobacterium ulcerans disease in patients who live in an endemic area. There are basically two presentations of M. ulcerans disease: non-ulcerative and ulcerative. Non-ulcerative forms present as:
Non-ulcerative forms • Papule: This is defined as a painless, raised skin lesion, less than 1 cm in diameter. The surrounding skin
is reddened (Fig. 2). This form is commonly seen in Australia.
• Nodule: A nodule is a lesion that extends from the skin into the subcutaneous tissue. It is 1–2 cm in diameter. It is usually painless but may be itchy and the surrounding skin may be discoloured compared to adjacent areas (Fig. 3). This form is commonly seen in Africa.
• Plaque: This is a firm, painless, elevated, well-demarcated lesion more than 2 cm in diameter with irregular edges. The skin over the lesion is often reddened or otherwise discoloured (Fig. 4).
• Oedematous form: There is a diffuse, extensive, usually non-pitting swelling. The affected area has ill-defined margins, is firm and painless and involves part or all of a limb or other part of the body. There may be colour changes over the affected region (Fig. 5a and 5b) and the disease may be accompanied by fever.
What you should know
Papule Nodule Plaque Oedematous form Figure 2 Figure 3 Figure 4 Figure 5a Figure 5b
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Clinical diagnosis
Ulcerative forms When fully developed, the ulcer has undermined edges and is indurated peripherally. The floor of the ulcer may have a white cotton wool-like appearance from the necrotic slough (Fig. 6a–d).
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The ulcer is usually painless, unless there is secondary bacterial infection. When there is more than one ulcer and the ulcers are close together, they often communicate beneath intact skin.
Figure 6a Hand
Figure 6c Back
Figure 6d Forearm
Figure 6b Leg
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Mycobacterium ulcerans osteomyelitis is initially painless, but subsequently frankly painful, and well localized. There is usually an identifiable area of increased warmth. A swelling then appears and this may progress to a fistula that discharges necrotic material. Incision of the swelling reveals gelatinous tissue and, beneath this, the bone has a moth-eaten appearance. Unlike open (contiguous) osteitis, the bone is the site of necrosis to a variable extent, similar to that seen in tuberculous osteomyelitis (Fig. 7).
• Reactive osteitis: Reactive (contiguous) osteitis occurs as a consequence of deep destruction of overlying soft tissues. Occasionally, the bone is exposed to the point of devascularization, necrosis of cortical bone, sequestration, and osteomyelitis. The macroscopic appearance is then that of white dead bone of almost normal appearance and texture.
Bone involvement • Osteomyelitis: This is true osteomyelitis. It may be focal or multifocal. The overlying skin is often intact with
no obvious lesion. Osteomyelitis may occur as a primary condition or as a metastatic condition, sometimes at a distance from a cutaneous lesion(s) or after a cutaneous lesion has healed.
Figure 7 Osteomyelitis – Leg
Complications and sequelae • Contractures
Contractures result from scarring caused by lesions over or close to joints (Fig. 8a and 8b). Ankyloses may follow.
• Bleeding There may be continuous minor bleeding or a sudden major haemorrhage. One must be careful to avoid large blood vessels beneath a lesion.
• Secondary infection Secondary bacterial infection may be caused by organisms such as staphylococci, streptococci, Pseudomonas sp., Corynebacterium sp., etc. Secondary infection may progress to cellulitis and septicaemia.
• Extension to deep structures Infection may extend beneath the deep fascia to involve tendon sheaths, muscle, blood vessels, nerves, bone and joints or may destroy periorbital tissue with loss of the eye.
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Clinical diagnosis
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• Other sequelae Hypertrophic scars and keloids may develop at infection and surgical sites including skin graft donor sites (Fig. 9). Squamous cell carcinoma (Marjolin’s ulcer) may appear in an unstable scar or persistent ulcer many years after initial infection with M. ulcerans. (Fig. 10).
Figure 9 Hypertrophic scar
Clinical diagnosis
Differential diagnosis The differential diagnosis of nodules is more difficult than that of ulcers. Some common differential diagnoses are described in Buruli Ulcer: Mycobacterium ulcerans infection (ref. WHO/CDS/CPE/GBUI/2000.1). Table 1 and figures 11a and 11b relate to differential diagnosis.
Figure 11a Figure 11b Leishmaniasis Tropical phagedenic ulcer
5
Papule
Psoriasis
Pityriasis
Note: Infection caused by other mycobacterial organisms can be mistaken for any of the above.
Nodule
Cyst
Lipoma
Onchocercoma
Boil
Lymphadenitis
Mycosis
Plaque
Leprosy
Cellulitis
Mycosis
Psoriasis
Haematoma
Table 1 Differential diagnoses of various forms of Buruli ulcer
Clinical diagnosis
14
Key points 1) Buruli ulcer disease presents as: papules, nodules, plaques, oedematous forms, ulcers
and bone infections. 2) Contractures are easier to prevent than to correct. 3) Osteomyelitis may arise when an ulcer invades bone or when infection is blood-borne.
Notes
Handling clinical specimens I Laboratory disinfection I Record-keeping
Chapter 2
Credit: WHO
Biosafety & record- keeping in laboratory
16
Biosafety and record-keeping in the laboratory
Handling clinical specimens Mycobacterium ulcerans is an environmental pathogen, and there are very few reports of person to person transmission. Nevertheless, precautions must be taken in the laboratory. Although transmission of M. ulcerans to laboratory workers has not been reported, it is possible that specimens may contain other unsuspected pathogens, in particular M. tuberculosis, hepatitis B virus, and HIV. Basic safety standards must always be followed: gloves, gowns and use of biosafety facilities (BSL 2 or 3) whenever possible. Care must also be taken to limit the formation of aerosols.
Laboratory disinfection Disinfection of nondisposable equipment and laboratory surfaces contaminated with mycobacteria requires special procedures, which differ from those used for viruses such as HIV, and other microorganisms. The use of quaternary ammonium compounds and sodium hypochlorite is discouraged: the former is ineffective and the latter is often used at suboptimal concentrations. Common antiseptics such as chlorhexidine gluconate and benzalkonium chloride exhibit no antimycobacterial activity even after treatment for 2 hours. The recommended products are indicated in Table 2. Besides the concentration of the disinfectant, time of contact is also important and must be at least 30 minutes.
This chapter will assist you to understand biosafety and record-keeping when dealing with Mycobacterium ulcerans disease.
O b j e c t i v e s
1
2
Biosafety & record- keeping in laboratory
Record-keeping Good quality laboratory records that make it possible to track each specimen through the laboratory are essential. This is best done with “bench books”. M. ulcerans is slow growing; therefore interim reports should be issued when available. For example, when Ziehl-Neelsen (ZN) or PCR has been performed, an interim report stating the result should be issued. When culture is first positive and a presumptive identification can be performed, it may be appropriate to issue a further preliminary report before finalizing the laboratory investigation of that specimen. Sample laboratory request and report forms can be found in Annexes 2 and 3.
Table 2 Disinfectants recommended for use in laboratories studying M. ulcerans
Disinfectant
Iodine
Notes
Collection & transport of clinical specimens
Procedures appropriate for different care-levels I Types of clinical specimens I Storage and transport of specimens
Collection and transport of clinical specimens
Chapter 3
20
All specimens, except swabs, must be obtained from surgically excised tissue taken in an operating theatre. Punch biopsies should never be collected in the field as they often are not diagnostic and may exacerbate the disease, promote secondary infection and delay definitive treatment. In specific situations, incisional biopsies in hospital may be performed to exclude other causes of skin lesions.
Procedures appropriate for different care-levels Generally, in each country there are three care-levels: peripheral (health centres and dispensaries), intermediate (general hospitals and district hospitals) and central (university teaching hospitals, regional hospitals and research centres). Specimen collection and laboratory procedures that can be performed at each level are outlined in table 3.
Collection & transport of clinical specimens
Collection and transport of clinical specimens
This chapter will assist you to collect the right clinical specimens and to transport them under appropriate conditions to the laboratory.
O b j e c t i v e s
Table 3 Specimen collection and laboratory methods for each care-level
Tests & Procedures
Surgery
Types of clinical specimens • Non-ulcerative forms
Specimens for laboratory confirmation from non-ulcerative forms (i.e., papules, nodules, plaques and oedematous forms—see Chapter 2) should be taken from the centre of the surgically excised tissue and should include the entire thickness of clinically-infected tissue.
Especially for non-ulcerative plaques and oedematous forms, the patient or the patient’s relative should be asked to indicate the site at which the lesion first appeared, as this is the most likely site to yield a positive diagnosis but several further biopsies should be taken from other parts of the lesion. Tissue fragments from the periphery of a lesion are not recommended for microbiological studies, because M. ulcerans is often not found here but such specimens may be most suitable for histopathology.
• Ulcerative forms Multiple swabs should be taken from different sites, especially from beneath the undermined edges of lesions (Fig. 13). Do not swab the slough in the centre of an ulcer. Specimens that include all levels of the skin and subcutaneous tissue are most suitable for histopathological study.
Collection & transport of clinical specimens
Figure 13 Swabbing the undermined edges of a Buruli ulcer
Figure 12 Specimen collection containers
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• Bone Diagnostic procedures to assess bone involvement should only be performed at centres providing intermediate and high-level services. For amputation specimens, the involved whole bone or curetted bone samples are required; when amputation is not necessary, curetted bone samples are appropriate.
Storage and transport of specimens Sample to be stored for immediate analysis—place in a sterile container without any additives.
Sample to be transported: • Analysis within 24 hours—keep the sample cool (ideally at 4 ºC), e.g. in an insulated container with a frozen cooling block. • Analysis after 24 hours:
– when refrigeration facilities are available, keep at 4 ºC—do not freeze; – when refrigeration facilities are not available, transport medium is essential. Liquid Middlebrook 7H9 broth
supplemented with polymyxin B, amphotericin B, nalidixic acid, trimethoprim and azlocillin (PANTA) is recommended. Supplementation with 0.5% agar achieves a semi-solid medium. (Specimens kept in transport medium may still be culture-positive up to 21 days.)
Transport for PCR analysis PCR is best performed directly on fresh tissue specimens prepared as described above. For ulcerative forms, dry cotton wool swabs stored in their plastic containers at ambient temperature are acceptable.
Notes
Specimens to collect I Direct examination I Culture I Antimicrobial susceptibility testing
Chapter 4
Credit: WHO
24
Specimens to collect Swabs: Use sterile cotton swab to collect pus or other exudates and place into either Amies or Stuarts Transport Medium for transport to the laboratory as early as possible. Tissue fragments: refer to Chapter 3.
Direct examination by microscopy Gram’s stain: Preparation of a Gram-stained smear is the method of choice for the identification of gram-positive, gram-negative bacteria and yeast cells. Wet mount: This is to detect fungal elements and yeast cells. It is prepared by mixing the sample with 15% potassium hydroxide (KOH) on a glass slide with a cover slip. It is then heated for 15 minutes to dissolve keratin. Examine at a magnification of x400 for fungal elements and yeast cells.
Secondary bacterial infection in Mycobacterium ulcerans disease
This chapter will assist you to diagnose secondary bacterial infection that may accompany Mycobacterium ulcerans disease.
O b j e c t i v e s
Secondary infection of Buruli ulcers is not as common as would be expected given the extent of skin loss. The reasons for this are unclear but may include an antibiotic effect of the M. ulcerans toxin, mycolactone. Despite this, secondary infections with Staphylococcus aureus and other bacterial pathogens are well known. They should be suspected when a lesion develops cellulitis, becomes painful or the patient becomes febrile.
1
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Culture Specimen should be processed as soon as they arrive in the laboratory. Choice of culture media depends on the result of the Gram-stain and wet mount studies. Bacteria: In case of secondary bacterial infection, all specimens (pus or exudate) should preferably be inoculated onto a minimum of three culture media.
Plates of blood agar: • Aerobic incubation at 35 °C for 18–20 hours for the isolation of staphylococci, streptococci, and Candida sp; • Anaerobic incubation at 35 °C for 48 hours for anaerobes such as Clostridium sp.
MacConkey or cystine-lactose-electrolyte-deficient (CLED) medium for the isolation of gram-negative rods. Incubation: at 35 °C for 18–20 hours for the identification of lactose and non-lactose fermenters. Robertson’s beef heart infusion broth may be inoculated as an enrichment broth and incubated 37 °C for 24 hours before sub-culturing on solid media.
Fungi: Culture on Sabouraud agar if fungal elements or yeast cells have been observed on the wet mount. Inoculated plates should be incubated at room temperature for at least 7 days.
Identification of cultured organisms: Pure subcultures of all bacteria or fungi isolated must be made and identified using standard methods. For example, coagulase test for staphylococci, bacitracin test and Lancefield grouping for streptococci species and biochemical tests for gram-negative bacilli.
Antimicrobial susceptibility testing Susceptibility to antimicrobial drugs differs from place to place and region to region. It is therefore necessary for local laboratories to determine their own susceptibility patterns. This will help in selecting the most appropriate antimicrobial agent for treatment. Ideally, antimicrobial susceptibility studies should be done on all isolates.
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4
Microbiological methods
Microbiological methods
Specimen preparation I Direct smear examination I Decontamination I In vitro culture I Identification
Chapter 5
Credit: WHO
Microbiological methods
Specimen preparation Tissue specimens • Dice tissue into small pieces in phosphate-buffered saline (PBS) or normal saline with a sterile single-use
or autoclavable scalpel blade. (Note: If equipment is to be re-used, it must first be placed in a disinfectant, then carefully brushed before sterilization to prevent cross-contamination, especially for PCR studies. See Table 2 for recommended disinfectants).
• Mix well (e.g. with a vortex mixer). • The specimen may also be prepared by grinding with a sterile mortar and pestle or Potter grinder.
Again, care should be taken to prevent cross-contamination when cleaning these instruments.
Swabs • Suspend swabs in a small volume of PBS or normal saline and then vortex well (e.g. 20 ml sterile tube). • Liquid transport medium containing swab may also be shaken (vortex) directly.
Microbiological methods for diagnosis of Mycobacterium ulcerans disease
This chapter will assist you to understand the various microbiological methods for the diagnosis of Mycobacterium ulcerans disease.
O b j e c t i v e s
A flow chart for laboratory diagnosis is presented in Annex 1.
1
Microbiological methods
Direct smear examination There are several staining techniques for mycobacteria: Ziehl-Neelsen (ZN), Kinyoun and auramine-rhodamine. The method used locally for the diagnosis of tuberculosis is applicable to M. ulcerans. In most cases this will be the ZN stain method (see Annex 5). The quantitation of smears should be in accordance with the method used locally for microbiological diagnosis of tuberculosis.
Decontamination prior to culture All specimens for primary isolation of M. ulcerans may contain contaminating microorganisms. Decontamination is necessary before attempting culture. The best results will be obtained from fresh specimens that are prepared and decontaminated immediately. Problems with bacterial or fungal overgrowth and loss of viable mycobacteria increase as the storage and transport time increase.
In vitro methods Several methods have been described to decontaminate specimens prior to culture for mycobacteria. Overly strong decontamination procedures will reduce the likelihood of obtaining a positive culture for M. ulcerans. The method of choice depends on the culture medium to be used: • for liquid culture (BACTEC), N-acetyl-L-cysteine-sodium hydroxide or the Petroff method is recommended
(see Annex 7); • for L-J medium, any of the described methods for decontamination of mycobacteria specimens is recommended
(see Annex 7). To control for excessive decontamination in the laboratory, which will reduce the yield of positive cultures, an overall rate of contamination in the range of 2–5% of all cultures is acceptable. The method of choice is therefore at the discretion of the microbiologist and will depend on types of specimens and degree of contamination.
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In vitro culture Culture media For solid media, L-J is the most suitable medium for M. ulcerans. For the BACTEC system, Middlebrook 7H12B medium is recommended (see Annex 8).
Culture conditions Mycobacterium ulcerans grows under the same conditions as M. tuberculosis except that the optimal temperature is 29–33 ºC. In liquid media (e.g. in the BACTEC system) M. ulcerans may also show enhanced growth under micro- aerophilic conditions (2.5–5% oxygen).
Culture duration Primary cultures are usually positive within 6–12 weeks incubation at 29–33 °C, but much longer incubation of up to 9 months may be necessary for some isolates. Duration of the incubation period should be selected according to the objective of the laboratory investigation.
Figure 14 Inoculation of M. ulcerans
into the tail of a mouse Note the swelling and ulcer
Mouse inoculation 1. Specimen preparation • Swabs Swirl the swab in a tube containing 1 ml sterile saline (0.85%). Mix well. Sample with sterile insulin syringe.
• Tissue fragments Grind tissue fragments in a tissue grinder (Potter) with saline (2 ml). Remove the supernatant into a sterile tube and sample with a sterile insulin syringe.
2. Animal inoculation Foot pad inoculation: 0.03 ml in the hind foot pad. Tail inoculation: 0.1 ml subcutaneously in the tail skin. Animals are observed weekly for inflammation near the site of injection. The infected tissue is sampled, decontaminated and inoculated in culture media.
In vivo methods Laboratory animals (usually mice) may be used for the primary isolation of M. ulcerans from patient specimens to diminish problems with contamination, and may offer improved sensitivity compared to in vitro methods. For foot pads, a 30 µl sample—prepared as described in the section on tissue specimens above—is injected subcutaneously (100 µl if tail injection is used, see Fig. 14). At the first sign of swelling or distress, the mice are sacrificed, and foot pad or tail biopsies collected under sterile conditions are then prepared for culture as for a routine specimen, including decontamination.
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Identification Positive primary cultures When primary cultures are positive, colonies suggestive of M. ulcerans appear yellowish, rough and have well-demarcated edges. African strains are more yellowish (Fig. 15a) than Australian strains (Fig. 15b), which may be only slightly pigmented.
A single, typical colony should be selected for subculture onto L-J medium. When BACTEC is positive, L-J is inoculated from the BACTEC medium.
Identification of subculture Growth rate Mycobacterium sp. are classified as either slow or rapid-growers. This distinction is based on whether isolated colonies are observed before or after 7 days on a solid medium. Isolated colonies are observed after solid media are inoculated with a 10-4 dilution of a standard culture suspension prepared at an optical density at 580 nm of 0.25, in a tube with a diameter of 2 cm. This corresponds roughly to a suspension containing 1 mg wet weight of bacilli per ml. A further distinction is that rapidly growing, but not slowly growing species are able to develop on simple media such as nutrient agar or peptone agar.
Microbiological methods
Figure 15a M. ulcerans isolates from Africa
cultivated on L-J medium; the isolates produce a light yellow pigment
Figure 15b M. ulcerans isolate from Australia cultivated
on L-J medium. The isolate does not produce pigment (nonchromogenic mycobacterium)
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Specific identification of M. ulcerans Phenotypic tests Mycobacterium ulcerans belongs to the slow-growing group. Tests for identification of M. ulcerans and related species are summarized in Table 4. The procedures for identification of slow growing mycobacteria are indicated in Annex 9. Specific phenotypic tests for the identification of M. ulcerans and related species are shown in Table 4. Differences between various subgroups according to their geographic origins are shown in Table 5. Drug susceptibility tests may be conducted for further identification. M. ulcerans is reliably resistant to isoniazid, para-aminosalicylic acid (PAS) and ethambutol but sensitive to rifampicin, streptomycin and several second line antituberculous drugs (see Table 6).
Table 4 Phenotypic characteristics of M. ulcerans and related species
Microbiological methods
33
Table 5 Characteristics of the different geographical subgroups of M. ulcerans
Microbiological methods
34
Table 6 In vitro susceptibility of M. ulcerans to antimycobacterial drugs
35
Identification of colonies by PCR Positive cultures may also be identified by PCR as described below and in Annex 10.
Identification of M. ulcerans by PCR Identification of M. ulcerans by PCR may be performed directly from clinical specimens or from culture media. Although there are several published methods, currently the best method is IS2404 PCR. However, PCR is relatively expensive and is notorious for producing false-positive results in laboratories which lack experience with this technology. Suitable clinical specimens for PCR include dry swabs, fresh tissue or specimens kept in transport medium. Specimens should be prepared as for culture, although decontamination is not necessary. Great care must be taken to keep the sample preparation, PCR master-mix preparation and agarose gel areas of the laboratory separate to prevent cross-contamination. It is advisable to include multiple negative controls in every PCR run. All results must be discarded if any negative control is positive. To control for inhibition, each PCR reaction is performed in duplicate. The second tube is “spiked” with approximately 100 molecules of purified M. ulcerans DNA. If this spiked positive control tests negative, the PCR reaction is being inhibited. Inhibition in clinical specimens can often be overcome by repeating the PCR using a 1:10 dilution of the extracted DNA sample.
PCR products are detected by ultraviolet transillumination of ethidium stained agarose gels. Presumed positive PCR results can be checked by Southern blot using an internal probe based on IS2404. With experience, it is acceptable to rely on comparison of the test sample with the positive control of the gel. If the two PCR products (positive control and test sample) align precisely, and the negative controls are negative, it can be concluded that the test sample is positive for M. ulcerans. Quality control measures must be in place. PCR results should be compared with culture results to monitor accuracy.
Microbiological methods
36
Sample results are illustrated in figure 16. It is recommended that Southern blotting or an equivalent method of verification be used to establish that the PCR product is the correct sequence when new laboratories are establishing M. ulcerans PCR.
The main advantage of PCR is that M. ulcerans disease can be definitively diagnosed within 24 hours of receipt of a clinical specimen by the laboratory. Culture confirmation takes 6 or more weeks. It is recommended that at present PCR be used as a rapid ancillary test and not as a replacement for culture and histology. In summary, the diagnostic PCR protocol consists of 4 phases: • Heat and alkaline lysis (to release DNA from M. ulcerans cells) • Extraction of total DNA from sample • PCR reaction to detect M. ulcerans-specific DNA in extracted total DNA
(primers slightly modified from Ross et al., 1997a) • Identification of PCR product (e.g. agarose gel electrophoresis) The full protocol is shown in Annex 10.
Microbiological methods
Figure 16 Electrophoresis gel under UV illumination ethidium bromide stained electrophoresis gel under UV light.
Lane 1-positive control; Lane 2,4,6: swab from patients with M. ulcerans infection; Lane 3 negative control; lane 5
swab from patient with chronic ulcer (non-M. ulcerans)
1 2 3 4 5 6
Histopathological methods
Histopathological methods
Selection of site for biopsy specimen I Fixation of tissue I Preparation of histopathological sections Gross changes I Histopathological changes
Chapter 6
Credit: WHO
38 A detailed history and description of the lesion that has been excised is very important
for a meaningful evaluation and for archival purposes. Name, age, sex, laboratory or hospital number and site of lesion are absolutely essential.
Selection of site for biopsy specimen Excisional specimens are advised. Specimens taken by punch are often unsatisfactory.
Non-ulcerative lesions Specimens should be obtained from the presumed centre of the lesion and include all levels of the skin and subcutaneous tissue down to fascia.
Ulcerative lesions Specimens should be taken from the edge of the ulcer and include the entire thickness of the skin and subcutis down to fascia.
Histopathological methods
Histopathological methods for diagnosis of Mycobacterium ulcerans disease
This chapter will assist you to understand the various histopathological methods for the diagnosis of Mycobacterium ulcerans disease.
O b j e c t i v e s
What you should know
1
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Fixation of tissue Optimally, the tissue should be fixed in neutral or buffered 10% formalin (pH 7.4). Ideally, the tissue should be fixed in a volume of formalin 10 times the volume of tissue for at least 24 hours before shipping. After fixation, the tissue can be shipped in smaller volumes of fixative. Care should be taken to identify the tissue with permanent markings on the container label. Bone must be decalcified before sectioning.
Preparation of histopathological sections Routine processing of fixed tissue is sufficient. Sections should be cut at 4-5 microns and stained by: 1) haematoxylin and eosin; 2) Ziehl-Neelsen for AFB; 3) Grocott methenamine-silver for fungi; and 4) tissue Gram’s stain for other bacteria (see Annex 6). Other stains are employed as indicated.
Gross changes Surface changes of non-ulcerated lesions often show loss of topographic markings and discolouration. Cut sections show changes in colouration, necrosis and mineralization. Lymph nodes show soft greyish-tan cut surfaces. After decalcification, cut sections of bone show yellowish necrosis of the marrow and often, thinning of the cortex.
Histopathological methods
Histopathological changes Skin changes Necrotic (active) stage: non-ulcerated lesions
The epidermis is intact, but is often hyperplastic. The upper dermis is usually intact but may show various stages of degeneration with infiltration of small numbers of inflammatory cells. There is contiguous coagulation necrosis of the lower dermis, subcutaneous tissue and underlying fascia (Fig. 17–19). There is oedema with remarkably few inflammatory cells, unless the lesion is infected secondarily by pyogenic bacteria. Adipose cells swell, but may lose their nuclei and retain their cell wall (fat cell ghosts—Fig. 20).
Histopathological methods
Figure 17 Section of surgically resected nodule of M. ulcerans disease. The central whitish area represents coagulation necrosis
Figure 18 Microscopic section of a nodule.
Note the massive coagulation necrosis of the lower dermis
and subcutaneous tissue. H & E x2
Figure 19 Skin and subcutaneous tissue from centre of a non-ulcerated widely disseminated lesion of M. ulcerans infection that covered 50 percent of the abdomen of a nine year old boy. Epidermis is intact. There is massive contiguous coagulation necrosis of the entire specimen. H & E x2
Figure 20 Necrotic base of Buruli ulcer showing many fat cell ghosts (upper portion) and many AFB (lower portion). ZN stain x50
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Most bacilli are in the deeper areas of the specimen but may invade the interstitium of the adipose tissue and lobular septa of the subcutaneous tissue (Fig. 24). Continuing necrosis of the dermis usually leads to degeneration of the epidermis and ultimate ulceration. Necrosis, however, may spread laterally with proliferation of AFB in the subcutaneous tissue and fascia (Fig. 25). Ulceration of the epidermis in such cases is often a very late event. The spread of disease in this manner leads to the plaque and oedematous forms of the disease.
Vasculitis is common in the subcutaneous tissue, often with occlusion of vessels by thrombi (Fig. 21 and Fig. 22). Varying degrees of mineralization are seen, especially in African patients. The ZN stain classically reveals large numbers of extracellular acid-fast bacilli (AFB); often in clusters and confined to the necrotic areas (Fig. 23).
Figure 21 Severe vasculitis in subcutaneous tissue of lesion of Buruli ulcer. Movat x80
Figure 22 Fat cell ghosts and vasculitis.
H & E stain x50
Figure 24 Subcutaneous tissue from the edge
of a Buruli ulcer showing fat cell ghosts with AFB in the interstitium.
ZN stain x100
Figure 23 ZN stain of a section parallel to that of Figure 18 shows AFB restricted to the centre of the lesion. Necrosis extends far beyond the focus of AFB x2
Histopathological methods
42 Figure 27 Subcutaneous tissue from margin of a Buruli ulcer showing necrosis and thickening of an interlobular septum. Septum contains masses of AFB. ZN stain x50
Figure 28 Early healing of a Buruli ulcer in the
organizing phase: lymphocytes, epithelioid and giant cells. H & E x50
Figure 30 Advanced stage of healing
Buruli ulcer showing scarring over most of the section.
H & E x25
Figure 29 Well formed delayed hypersensitivity granuloma in healing Buruli ulcer. H & E x50
Figure 25 Masses of AFB infiltrate the base of the edge of a Buruli ulcer. AFB typically in clusters. ZN x100
Figure 26 Biopsy specimen from the edge of a
Buruli ulcer showing undermining of the dermis and massive necrosis of the
skin, dermis, subcutis and the fascia
Histopathological methods
43
Necrotic (active) stage: ulcerative lesions Ulcers are undermined with reepithelialization of the edges of the lesion and undersurface of the overlying flap of the dermis (Fig. 26). Adjacent epidermis is usually hyperplastic. The base of the pristine ulcer contains a necrotic slough of cellular debris and fibrin, sometimes with a central eschar. There is contiguous coagulation necrosis of the subcutaneous tissue and fascia similar to that described for non-ulcerated lesions (Fig. 25 and Fig. 27). AFB are located in the base of the central slough and necrotic subcutaneous tissue. The disease rarely extends into underlying muscle. Vasculitis and mineralization are seen often (Fig. 21).
Organizing (early granulomatous stage) Early healing is characterized by a poorly organized granulomatous response in the dermis and subcutaneous tissue (Fig. 28). The granulomatous infiltration comprises swollen macrophages (epithelioid cells), Langhans’ giant cells and lymphocytes. These eventually form organized tuberculoid granulomas. Foamy macrophages, lymphocytes and plasma cells are sometimes seen at the margin of necrotic fat. AFB are scarce or absent.
Healing stage As healing advances, granulation tissue forms followed by fibrosis and a depressed scar (Fig. 29 and Fig. 30). AFB are seldom seen.
Lymph nodes Although clinical lymphadenopathy is rarely appreciated, significant lymphadenitis is often seen histopathologically, both in lymph nodes adjacent to lesions and in regional nodes. Those adjacent to lesions may show marked invasion of the capsule by AFB (Fig. 31). The parenchyma is often markedly necrotic with destruction of cortical lymphoid tissue (Fig. 32). In such cases the entire node may be invaded by AFB. Regional lymph nodes, however, may show only sinus histiocytosis. Granulomatous changes are usually not seen, and AFB are rarely seen in regional nodes.
Histopathological methods
44
Figure 33 X-ray of the leg showing destruction of the bone. Note: the patient had a Buruli ulcer over the affected area
Figure 34 Osteomyelitis of tibia showing
necrosis of the marrow and erosion of trabeculae. H & E stain x2.5
Figure 35 Osteomyelitis of tibia with masses of AFB in necrotic marrow. ZN stain x50
Figure 31 Lymphadenopathy in Buruli ulcer. The parenchyma of the node is necrotic and the capsule is heavily infiltrated by AFB. ZN stain x100
Figure 32 Necrotic lymphadenitis in a lymph
node proximal to Buruli ulcer. The medulla is destroyed and only remnants of the cortical lymphoid tissue remain. ZN stained parallel
sections showed large numbers of AFB. H & E stain x5
Figure 36 Osteomyelitis of tibia showing
necrosis of marrow and a trabecula of bone undergoing dissolution in
area of AFB. ZN stain x100
Histopathological methods
Bone changes
Bone may be affected by direct extension from an overlying lesion of Buruli ulcer, or at a site distant from recognized lesions, presumably by haematogenous spread of M. ulcerans (Fig. 33). Histopathologically, the marrow is extensively necrotic and the bone trabeculae are eroded (Fig. 34). AFB are present in varying numbers, most often in the necrotic marrow (Fig. 35 and Fig. 36). Although some lesions in bone seem to be purely an effect of the M. ulcerans in the bone, approximately 50% of the osteomyelitic lesions are coinfected by pyogenic organisms such as streptococci, staphylococci and Corynebacterium sp. In such instances, there is suppuration and the organisms may be visible in Gram’s stained sections. Well formed granulomas may develop producing a chronic osteomyelitis that is probably caused by M. ulcerans.
Patients with extensive disease (comment) Patients with aggressive oedematous lesions involving large body areas often have widespread oedema and impaired renal function, or other evidence suggesting visceral organ involvement. Such patients sometimes die early in the course of the disease. While some authorities suspect that these events are attributable to a systemic effect of the toxin, this question can only be resolved by increased efforts to study the pathophysiology of such patients and by the study of autopsy specimens.
Histopathological methods
46
As with all laboratory tests, the quality of the results produced depends on the quality and prompt delivery of the samples. It is not advisable to conclude that a patient does not have Buruli ulcer even if all tests are negative. Such situations may arise if biopsies or swabs are taken from areas where no organisms are present, or transport times are prolonged. For example, in one series, 500 patients were considered to have proven Buruli ulcer using a strict definition of having at least two of the following tests positive: culture, PCR, histology or ZN. When each diagnostic method was considered alone, the following sensitivities were obtained: ZN 40–80%, culture 20–60%, histology > 90%, PCR > 90%. ZN and culture in particular are dependent on the type of clinical lesion. For example ZN sensitivity for nodules was 40%, for ulcers 60% and plaques 80%. For culture, bone specimens were positive in only 20% of cases, 50% for ulcers and 60% for plaques. Sensitivity of culture may be further improved by initial passage in mice (up to 75%). Clearly it is not advisable to exclude the diagnosis or to conclude that the patient has Buruli ulcer based on any one laboratory test. Although uncommon, false positive ZN or PCR results or even false positive culture results have occurred. If the result from the laboratory does not fit with the clinical presentation, or is questioned by the clinician, laboratory tests should be repeated on freshly collected specimens. Ideally, results obtained using several modalities and multiple samples should be considered together. However, in practice, in endemic areas, experienced clinicians commonly make accurate presumptive diagnosis on clinical grounds alone, or by using a combination of clinical appearances and a ZN stained smear.
Notes on interpretation of laboratory tests
Annexes
Flow-chart for the laboratory diagnosis I Laboratory request form I Laboratory report form I Preparation of culture media Microbiological staining techniques I Histopathological staining techniques I Decontamination methods I Mycobacterium
ulcerans culture with BACTEC 460 TB instrument I Biochemical and culture tests used for identification of slow-growing mycobacteria I PCR protocol I Manufacturers’ addresses I Work of WHO on Buruli ulcer I Some research institutions involved in Buruli ulcer activities I Some NGOs and others involved in Buruli ulcer activities I Members of the WHO
Advisory Group on Buruli ulcer I Suggested reading
Annexes
1 swab
1. Name of institution, address . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2. Subdistrict . . . . . . . . . . . . . . . . . . . . . . . . . District . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Region . . . . . . . . . . . . . . . . . . . . . . . . . . Country . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3. Name of officer completing the form (last/first) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. Title . . . . . . . . . . . . . . . . . . . . . . . Speciality . . . . . . . . . . . . . . . . . . . . . . .
B. Patient information Patient identification number . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5. Health facility ID number . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Date of admission (dd/mm/yy) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6. Name (last/first) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7. Age . . . . . . . . . (months/years ) 8. Sex M F
9. Address . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
10. Occupation of patient . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
11. Brief description of the lesion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12. Site of first lesion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
13. List family contacts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
14. Patient classification New case Recurrent case Same site Different site
C. Location of lesion(s)
Lower limb: Right Left Thorax Head and neck
D. Clinical form
16. Unifocal forms
Inactive: Scar Amputation Others, specify . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Annexes 1–5
50
17. Multifocal forms (please indicate the location of each clinical form) Location of lesion(s) Clinical form
a. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
b. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
c. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
d. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
e. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
f. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Institutional information
c. Name of officer completing the form (last/first) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
d. Title . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Speciality . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Specimen laboratory number
F. Tests requested ZN Culture PCR Histopathology
Received in laboratory Date (dd/mm/yy) . . . . . . . . . . . . . . . . . . . . . . . . . . . . Time . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Name of officer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Signature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
51
Laboratory report form3 Tests summary ZN Culture PCR on specimen Histopathology
Positive
Negative
Culture
Positive Time from inoculation to first positive primary culture (in weeks)
Negative Species isolated . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
PCR on specimen
52
Annexes 1–5
Preparation of culture media Löwenstein-Jensen (L-J) medium There are three groups of components which are prepared separately and then added to make the medium: 1. Mineral solution 2. Malachite green solution 3. Homogenized whole eggs
• Mineral solution – potassium dihydrogen phosphate anhydrous (KH2PO4) 2.40 g – magnesium sulphate (MgSO4.7H2O) 0.24 g – magnesium citrate 0.60 g – asparagine 3.60 g – glycerol (reagent grade) 12 ml – distilled water 600 ml
Dissolve the ingredients following the order above, in distilled water, by heating. Autoclave at 121 °C for 30 minutes to sterilize. Cool to room temperature. This solution may be kept indefinitely and may be stored in suitable amounts in the refrigerator.
• Malachite green solution 2% – malachite green dye 2 g – sterile distilled water 100 ml
Using aseptic techniques, dissolve the dye in sterile distilled water by placing the solution in an incubator for 1 to 2 hours. This solution cannot be stored indefinitely and may precipitate or change to less-deeply coloured solution. In either case, discard and prepare a fresh solution.
• Homogenized whole eggs Fresh hen’s eggs, not more than 7 days old, are cleaned by scrubbing thoroughly with a hand brush in warm water and a plain alkaline soap. Let the eggs soak for 30 minutes in soap solution. Rinse eggs thoroughly and soak them for 15 minutes in 70% ethanol. Remember to wash your hands before handling the clean, dry eggs. Crack the eggs with a sterile knife into a sterile flask and beat them with a sterile egg whisk or in a sterile blender.
Preparation of the complete medium – mineral solution 600 ml – malachite green solution 20 ml – homogenized eggs (20–25 eggs depending on the size) 1000 ml
4
53
Annexes 1–5
The complete egg medium is distributed in 6–8 ml volumes in sterile 14 or 28 ml McCartney bottles or in 20 ml volumes in 20x 150 mm screw-capped test tubes and the tops tightly closed. Inspissate the medium (see below) within 15 minutes of distribution to prevent sedimentation of the heavier ingredients.
Coagulation of medium (inspissation) Before loading, heat the inspissator to 80 °C to hasten the build up of the temperature. Place the bottles in a slanted position in the inspissator and coagulate the medium at 80–85 °C for 45 minutes. Do not reheat the medium. The quality of egg media deteriorates when coagulation is done at a too high temperature or for too long. Discolouring of the coagulated medium may be due to excessive temperature. The appearance of holes or bubbles on the surface of the medium also indicates faulty coagulation procedures. Discard poor quality media.
Sterility check After inspissation, the whole batch of media or a representative sample of culture bottles should be incubated at 35–37 °C for 24 hours as a sterility check.
Storage Eggs may sometimes contain antibiotics which inhibit the growth of mycobacteria. The origin of the eggs must be known to control their quality. The media should be dated, stored and may be kept in the refrigerator for several weeks with caps tightly closed to prevent the medium from drying out. For optimal isolation of mycobateria, L-J media should not be older than 4 weeks.
Middlebrook 7H10 and 7H11 agar medium Middlebrook 7H10 may be made from basic ingredients or prepared from commercially available 7H10 agar- powdered base and Middlebrook oleic acid-albumin-dextrose-catalase (OADC) enrichment. 7H11 is 7H10 agar enriched by the addition of an enzymatic digest of casein. It is best to prepare 7H10 and 7H11 medium in small quantities of 200–400 ml to minimize the heating needed to melt the agar. Boiling the basal medium before auto- claving (either to solubilize the agar or to provide stocks of prepared base that may be stored and boiled for later use) should be avoided because repeated heating compromises the quality of the medium. When Middlebrook 7H10 or 7H11 medium is used, it must be incubated under micro-aerophilic conditions (2.5– 5.0% oxygen). The exposure of Middlebrook 7H10 or 7H11 agar to either daylight or heat may result in the release of formaldehyde in sufficient concentration to inhibit the growth of mycobacteria.
54
Annexes 1–5
Microbiological staining techniques Ziehl-Neelsen (ZN) staining The reagents described here are strictly for use with the hot Ziehl-Neelsen method only. The hot method is superior to cold methods, such as the Kinyoun. Concentrations recommended are slightly different from what is often found in handbooks. The fuchsin concentration is slightly higher and the methylene blue lower, providing the best possible contrast (strong red bacilli with a light blue background). Other concentrations and cold methods may give satisfactory results under otherwise optimal conditions. However, when other conditions (microscope, light, technician training) are less well controlled, it is strongly recommended to use the concentrations given below and the hot method for a better colour contrast.
REAGENTS
• Fuchsin – basic fuchsin 10 g – 95% ethanol (technical grade) 100 ml – dissolve basic fuchsin in ethanol Solution 1
• Phenol – phenol crystals 5 g – distilled water 85 ml – dissolve phenol crystals in water Solution 2
Mix 10 ml of solution 1 with 90 ml of solution 2 and store in a tightly stoppered amber-coloured bottle. Label bottle with name of reagent and dates of preparation and expiry. Can be stored at room temperature for at least 12 months. Filter before or at the time of use.
• Decolourizing solution – concentrated hydrochloric acid 3 ml – 70% ethanol (technical grade) 97 ml
Carefully add concentrated hydrochloric acid to 70% ethanol. Always add acid slowly to alcohol, not vice versa. Store in an amber-coloured bottle. Label bottle with name of reagent carbolfuchsin and date of preparation. Can be stored at room temperature indefinitely.
5
55
Counterstain – methylene blue chloride 0.1 g – distilled water 100 ml Dissolve methylene blue chloride in distilled water in a tightly stoppered amber-coloured bottle. Label bottle with name of reagent and dates of preparation and expiry. Can be stored at room temperature for at least 12 months.
Procedure 1. Place the numbered slides on a staining rack in batches (maximum 12). Ensure that the slides do not touch
each other. 2. Flood entire smear with ZN carbolfuchsin which has been filtered prior to use; the most practical way is to
pour stain over the slide through a funnel equipped with filter-paper. 3. Heat the slide slowly until it is steaming for 3–5 minutes. Do not let the stain boil dry. 4. Rinse with gentle stream of running water until free stain is washed away. 5. Flood the slide with the decolourizing solution for 3 minutes. 6. Rinse the slide thoroughly with water. Drain excess water from the slide. 7. Repeat steps 5 and 6 if the smear is still too red. 8. Flood the slide with counterstain. 9. Allow the smear to counterstain, usually for a maximum of 60 seconds. If after repeated exposure to acid-
alcohol, the smear cannot be sufficiently discoloured, counterstain a bit longer. 10. Rinse the slide thoroughly with water. Drain excess water from the slide. 11. Allow smears to air-dry. Do not blot. Keep slide out of direct sunlight and read as soon as possible.
Figure 37 ZN stained smear from a Buruli ulcer
showing red extracellular AFB against a blue background
56
Quantitation scale
No. of AFB seen on average No. of fields to screen Report
No. of AFB / 100 immersion fields 100 No AFB observed 1–9 AFB / 100 immersion fields* 100 Record exact figure 10–99 AFB / 100 immersion fields 100 + 1–10 AFB / 1 immersion field 50 + + > 10 AFB / 1 immersion field 20 + + +
*A finding of three or fewer bacilli in 100 fields does not correlate well with culture positivity, but should be reported.
Fluorochrome staining REAGENTS
Auramine O – auramine powder 0.1 g – 95% ethanol (technical grade) 10 ml – dissolve auramine in ethanol Solution 1
Note: Auramine is carcinogenic, direct contact with skin should be avoided.
Phenol – phenol crystals 3.0 g – distilled water 87 ml – dissolve phenol crystals in water Solution 2
Mix solutions 1 and 2 and store in a tightly stoppered amber-coloured bottle away from heat and light. Do not use after 3 months. A precipitate usually forms but does not indicate deterioration; however, the solution should be filtered during the staining procedure.
Annexes 1–5
Decolourizing solution – concentrated hydrochloric acid 0.5 ml – 70% ethanol (technical grade) 100 ml
Carefully add concentrated hydrochloric acid to the ethanol. Always add acid slowly to alcohol, not vice versa. Store in amber-coloured bottle. Label bottle with name of reagent and date of preparation. Keeps indefinitely.
Counterstain – potassium permanganate (KMnO4) 0.5 g – distilled water 100 ml
Dissolve potassium permanganate in distilled water in a tightly stoppered amber-coloured bottle. Label bottle with name of reagent and dates of preparation and expiration. Store at room temperature for up to 3 months.
The potassium permanganate (0.5%) as described above tends to give a very dark background. This makes it difficult to keep the smear in focus. At lower concentrations this effect is less, however, such weak solutions of KMnO4 are unstable and not preferred by many laboratories.
Figure 38 Fluorochrome stained smear showing
AFB as bright yellow rods against a dark background
58
Procedure Prepare fairly thick smears from homogenized biopsy material. Such smears are easier to examine because of the more visible background.
1. Place numbered smears on a staining rack in batches (maximum 12). Ensure that the slides do not touch each other.
2. Flood entire smear with auramine O. Use a funnel equipped with a Whatman #1 filter-paper to pour the stain on the slides. Allow to stand for 15 minutes, making sure that the staining solution remains on the smears. Do not heat!
3. Rinse with water and drain. Distilled water is usually recommended but this is often not available in field laboratories. Experience from some laboratories has shown that use of tap water is always satisfactory. A suitable alternative would be dechlorinated water (i.e. water that has been exposed to air for 24 hours).
4. Decolourize with 0.5% acid-ethanol for 2 minutes. 5. Rinse with water and drain. 6. Flood smears with counterstain for 2 minutes. Time is critical because counterstaining for longer periods may
quench the fluorescence of AFB. 7. Rinse with water and drain. 8. Allow smears to air-dry. Do not blot. Read as soon as possible, keep slides in the dark (i.e. in a closed slide-box).
Quantification scale (see that for ZN staining)
Fluorescent microscopy magnification 200 or 250x 400x 630x
Number of AFB count Divide observed Divide observed Divide observed count by 10 count by 4 count by 2
To adjust for altered magnification of the fluorescent microscope, divide the number of organisms seen by the factor provided and refer to the quantification table for ZN smear for the appropriate value to report.
59
1. Harris’ haematoxylin and eosin (H & E) procedure (without mercury)
Note: This procedure stains tissue elements and bacteria more intensely than many other H & E procedures. Use of potassium permanganate in place of mercury salts is less hazardous to the environment and individuals.
The procedure is intended for specimens fixed in 10% buffered neutral formalin and tissue sections cut at 4-6 µm thick. Control tissue should contain nuclei, cytoplasmic structures, connective tissue and if possible bacteria.
SOLUTIONS • Harris’ hematoxylin – potassium or ammonium alum 100 g – distilled water 500 ml Dissolve with the aid of heat. In a separate container combine the following: – hematoxylin crystals 5 g – absolute ethanol 50 ml – distilled water 250 ml Dissolve (may be warmed) and add: – 0.25% potassium permanganate 250 ml Allow to stand 3 minutes stirring and combine this solution with the above alum solution. Cool in running water, and add 20 ml of glacial (100%) acetic acid. Filter before use.
• 1% acid-alcohol – 95% ethanol 736 ml – deionized water 263.2 ml – concentrated hydrochloric acid 10 ml
• Ammonia water – deionized water 1000 ml – 28% ammonium hydroxide 4 ml
6
60
• 1% Eosin stock solution – eosin Y, water-soluble 1 g – deionized water 100 ml
• 1% Phloxine stock solution – phloxine B 1 g – deionized water 100 ml
• Eosin—phloxine solution Combine the following: – eosin stock solution 100 ml – phloxine stock solution 10 ml – 95% ethanol 780 ml – glacial acetic acid 4 ml This solution is good for approximately one week.
Staining procedure 1. Deparaffinize slides and hydrate to water. 2. Stain in freshly filtered Harris haematoxylin for 10 minutes.
Figure 39
This section showing panniculitis is from a Buruli ulcer patient stained
by the H & E method
Note: the nuclei of cells are blue and the connective tissue is pink
61
Annexes 6 –11
3. Wash in warm running tap water for 5 minutes. 4. Dip twice in 1% acid-alcohol to differentiate. 5. Stop the differentiation by dipping in warm tap water and then dipping in weak ammonia water or saturated
lithium carbonate until section begins to turn bright blue. 6. Wash in warm running tap water for 10 minutes.
Note: if nuclear staining is weak, return to step 2. If the background is not clear return to step 4 but use only 1 quick dip in the acid alcohol. 7. Counterstain in eosin-phloxine for 2 minutes 8. Dehydrate and clear through 2 changes successively of 95% ethanol, absolute ethanol and xylene. Slides
should remain in each for 2 minutes. 9. Mount in a resinous mounting medium.
2. Ziehl-Neelsen (ZN) method for acid-fast organisms Adapted from technical SOP 5.23, Armed Forces Institute of Pathology (AFIP), Washington, DC, USA
Note: This technique is used to demonstrate acid-fast organisms other than Nocardia sp. and leprosy bacilli. The procedure is intended for specimens fixed in 10% buffered neutral formalin and sections cut at 4–6 micrometers thick. Control sections should contain known M. tuberculosis or M. ulcerans.
SOLUTIONS • ZN carbolfuchsin solution – phenol (fused crystal, melted) 25 ml – absolute ethanol 50 ml – basic fuchsin 5 g – deionized water 500 ml Store in a warm but open place to maintain the solution in liquid form.
• Acid-alcohol – 70% ethanol 100 ml – concentrated hydrochloric acid 1 ml
• Methylene blue solution (working) – methylene blue crystals 3 g – deionized water 600 ml
Annexes 6 –11
62
Staining procedures 1. Deparaffinize and hydrate to deionized water. 2. Stain in ZN carbolfuchsin for 30 minutes. Note: if organisms fail to stain, prepare new carbolfuchsin solution.
3. Wash in cool tap water for 10 minutes. Note: if tap water is chlorinated, wash for a shorter time.
4. Differentiate slides individually with acid-alcohol. 5. Wash in running water for 3 minutes. 6. Counterstain by dipping slide individually in working methylene blue solution then rinsing them in tap water. 7. Dehydrate and clear in 2 successive changes in 95% ethanol, 100% ethanol and xylene. 8. Mount in a resinous mounting medium:
• AFB: .....................................red • Background: .............blue
Figure 40 This section of a lymph node from a Buruli ulcer patient is stained by
the ZN method. The AFB are red and the background tissue is blue
63
Annexes 6 –11
3. Grocott’s method for fungi (GMS) Adapted from technical SOP 5.10, Armed Forces Institute of Pathology (AFIP), Washington, DC, USA
Note: This technique demonstrates all forms of fungi, however, Histoplasma capsulatum and Nocardia asteriodes may require extended time in the methenamine-silver solution. The procedure is intended for specimens fixed in 10% buffered neutral formalin and tissue sections cut at 4-6 µm thick. Control tissues must be from a known fungal infection containing fungal eleme